Novel 7-oxo-7-desacetylaminocolchicine compounds



United States Patent Claims priority, application France, June 19, 1963,938,630; Sept. 18, 1963, 947,899 Int. Cl. C07c 49/76 U.S. Cl. 260-590 4Claims ABSTRACT OF THE DISCLOSURE 7-oxo-7-desacetyl-amino-colchiciniccompounds of the formula wherein Z is selected from the group consistingof lower alkoxy amino, lower alkylamino, dilower alkylamino,aralkylamino and lower alkyl-aralkyl amino and to a novel process fortheir preparation. The numbering system is that of system RR] 3923 ofRing Index, 2nd ed., 1960. The invention further relates to novelanti-mitotic compositions and to a method of modifying cell mitosis.

. PRIOR APPLICATION This application is a divisional application ofcopending, commonly assigned US. patent application Ser. No. 374,168,filed June 10, 1964, now US. Patent No. 3,393,240.

The novel compounds of Formula I possess strong antimitotic activity incellular matter undergoing mitosis. They are useful industrially inagriculture for the modification of mitosis and the creation ofpolyploids either by drenching the cultivated soil with aqueoussolutions or suspensions of the compounds or by preliminary treatment ofthe seeds with the product per se or dilutions thereof in a solvent oron a support. The compounds are also useful for the preparation of othercolchicine compounds with known physiological properties.

OBJECTS OF THE INVENTION It is an object of the invention to provide thenovel 7- oxo-7-desacetylamino-colchicine compounds of Formula I.

It is another object of the invention to provide novel intermediates forthe 7-oxo-7-desacetylamino-colchicine compounds of Formula I.

It is a further object of the invention to provide a novel process forthe preparation of the 7-oxo-7-desacetylamino-colchicine compounds ofFormula I.

It is an additional object of the invention to provide novelanti-mitotic compositions.

It is another object of the invention to provide a novel method ofmodifying mitosis.

These and other objects and advantages of the inven- 'ice tion willbecome obvious from the following detailed description.

THE INVENTION The novel colchicine compounds of the invention have theformula wherein Z is lower alkoxy.

Examples of compounds within the scope of Formula I are 7-oxo-7-desacetylamino-colchicine,7-oxo-7-desacetylamino-colchiceinamide,7-oxo-7-desacetylamino-N-methyl-colchiceinamide,7-oxo-7-desacetylamino-N-benzyl-colchiceinamide,7-ox0-7-desacetylamino-N-ethyl-colchiceinamide,7-oxo-7-desacetylamino-N-propyl-colchiceinamide,7-oxo-7-desacetylamino-N-isopropyl-colchiceinamide,7-0x0-7-desacetylamino-N-butyl-colchiceinamide,7-oxo-7-desacetylamino-N,N-dimethyl-colchiceinamide,7-oxo-7-desacetylamino-N,N-diethyl-colchiceinamide,7-oxo-7-desacetylamino-N-methyl-N-ethyl-colchiceinamide, 7-oxo-7-desacetylamino-N-methy1-N-benzyl-colchiceinamide,7-oxo-7-desacetylamino-N-ethyl-N-isopropyl-colchiceinamide, etc.

The novel process of the invention for the preparation of the7-oxo-7-desacetylamino-colchicine compounds of Formula I comprisesreacting N-desacetyl-colchiceine with a chlorinating agent to formN-chloro-N-desacetylcolchiceine dehydrochlorinating the latter with analkaline agent to form 7-imino-7-desacetylamino-colchiceine, subjectingthe latter to acidic hydrolysis to form7-oxo-7-desa'cetyla-mino-colchiceine, reacting the latter with a diazolower alkane to form a mixture of 7-oxo-9-lower alkoxy-10-demethoxy-7-desacetylamino-colchicine and 7-oxo-l0- loweralkoxy-9-demethoxy 7 desacety1arnino-iso-colchicine, separating themixture into its components and reacting 7-oxo-10-loweralkoxy-10-demethoxy-7-desacetylamino-colchicine with a compound of theformula II wherein R and R are selected from the group consisting ofhydrogen, lower alkyl and aralkyl to form the corresponding7-oXo-7-desacetylamino-colchiceinamide. The reaction scheme isillustrated in Table I.

TABLE I 3 TABLE ICnli1zued CHaO CHaO CHaO -O CHaO -O CH CH3 CHzO whereinR is lower alkyl and R and R have the above definition.

It was completely unexpected that the chlorination ofN-desacetylcolchiceine would form a monochloro product and not apolychlorinated product. The said chlorination is preferably effectedwith N-chloro-succinimide in the presence of an acid such as aqueousacetic acid. This immediately forms N-chloro-N-desacetyl-colchiceinewhich crystallizes from solution without undergoing superfluousadditional chlorination.

The dehydrochlorination of N-chloro-N-desacetyl-colchiceine-js effectedwith a strong base such as an alkali metal hydroxide or carbonate,preferably in an alcoholic media such as methanol or ethanol.7-imino-7-desacetylamino-colchiceine is a relatively stable product ascompared ketimines in general and is obtained in good yields.

The hydrolysis of 7-imino-7-desacetylamino-colchiceine is easilyeffected in the presence of an organic acid such as aqueous acetic acid.The reaction of 7-oxo-7-desacetylamino-colchiceine with a diazo loweralkane such as diazomethane is preferably effected in an organic solventsuch as methylene chloride and/or methanol. The separation of thecolchicine and iso-colchicine compounds can be effected by fractionalcrystallization from an organic solvent such as methanol.

The reaction of 7-oxo-l0-lower alkoxy-lO-demethoxy-7-desacetylamino-colchicine with a nitrogen compound of Formula II ispreferably effected in an aqueous or lower alkanolic media at normaltemperature, such as room temperature. Examples of suitable compounds ofFormula II are ammonia; primary lower alkyl and phenyl lower alkylamines such as methylamine, benzyl amine, ethylamine, propyl amine,isopropyl amine, butyl amine, etc. and secondary lower alkyl and phenyllower alkyl amines such as dimethyl amine, diethylamine, methyl ethylamine, ethyl isopropyl amine, methyl benzyl amine,

etc. a

A preferred mode of the process of the invention for the preparation ofa 7-oxo-7-desacetylamino-colchicine .4 compound comprises reactingN-desacetyl-colchiceine with N-chloro-succinimide to formN-chloro-N-desacetylcolchiceine, dehydrochlorinating the latter with analkali metal hydroxide such as methanolic potassium hydroxide to form7-imino-7-desacetylamino-colchiceine, reacting the latter with aqueousacetic acid to form 7-oxo-7-desacetylamino-colchiceine, reacting thelatter with diazomethane to form a mixture of7-oxo-7-desacetylaminocolchicine and7-oxo-7-desacetylamino-isocolchicine, separating the two components byfractional crystallization and recovering7-oxo-7-desacetylaminc-colchicine which may be reacted further with anitrogen compound of Formula II if desired to form the corresponding7-oxo-7- desacetylamino-colchiceinamide.

In the following examples there are described several preferredembodiments to illustrate the invention. However, it should beunderstood that the invention is not intended to be limited to thespecific embodiments.

EXAMPLE I Preparation of 7-oxo-7-desacetylamino-colchicine StepA.Preparation of N-chloro-N-desacetyl-colchiceine: 20' gm. ofN-desacetyl-colchiceine were dissolved in 10 cc. of acetic acid and 200cc. of water while stirring for 5 minutes at ambient temperature. Then,a solution of 8.2 gm. of N-chloro-succinimide in 82 cc. of acetic acidwas added thereto at 60 C. and the reaction mixture was stirred for 15minutes at ambient temperature. The product was vacuum filtered, washedwith water and dried in vacuum to obtain 17.2 gm. ofN-desacetyl-N-chloro-colchiceine having a melting point of 170 to 174 C.and a specific rotation (c.=0.5% in chloroform) which was used as suchfor the following step of the synthesis.

The product occurred in the form of needles insoluble in water, aqueousdilute acids, ether and benzene, slightly soluble in alcohols andsoluble in acetone and chloroform.

This compound has not been described in the literature.

Step B.Preparation of 7-imino-7-desacetylamino-colchiceine: 17.2 gm. ofN-chloro-N-desacetyl-colchiceine were dissolved in 85 cc. of a 2 Nmethanolic solution of potassium hydroxide and the solution was allowedto stand for 17 hours at room temperature. Then water was added afterwhich acetic acid was added dropwise until a pH of 6.5 to 6.7 wasobtained. The product was cooled, vacuum filtered washed with water,triturated in acetone and dried to obtain 12.45 gm. of7-imino-7-desacetylamino-colchiceine having a melting point of C.(aqueous solvate), which was used as such for the following step of thesynthesis.

For analysis, the said product was recyrstallized in the methanol and amethanolic solvate having a melting point of C. was obtained, which whenrecrystallized in ethyl acetate, yielded a product melting at 178 C.This product occurred in the form of needles, insoluble in water, etherand benzene, slightly soluble in acetone, soluble in aqueous diluteacids and alkalis, alcohols and chloroform.

Analysis (C H O N): Molecular weight=34l.35. Calculated: C, 66.85%; H,5.61%; N, 4.10%; O, 23.44%. Found: C, 66.8%; H, 5.7%; N, 3.80%; O,23.7%.

This compound is not described in the literature.

Step C.Preparation of 7-oxo-7-desacetylamino-colchiceine: 12.45 gm. of7-imino-7-desacetylamino-colchh ceine were dissolved in 125 cc. ofdiluted 50% acetic acid and the solution was heated for 30 minutes at 50C. and 125 cc. of water were slowly added thereto. After scratching, the7-oxo-7-desacetylamino-colchiceine crystallized from the media. Theproduct was cooled, vacuum filtered, washed with water and dried toobtain 8.7 gm. of 7-oxo-7-desacetylamino-colchiceine having a meltingpoint of 154 C. used without further purification for the next step ofthe process.

The said product after being recrystallized from a mixture of methylenechloride and ethyl acetate, had a melting point of 157 C. and occurredin the form of little rods insoluble in water, in aqueous dilute acidsand alkalis, ether and benzene, slightly soluble in acetone and solublein alcohols and chloroform.

Analysis (C H O Molecular weight=342.33. Calculated: C, 66.66%; H,5.30%. Found: C, 66.6%; H, 5.4%.

This compound is not described in the literature.

Step D.-Preparation of 7-oxo-7-desacetylamino-colchicine: 9.2 gm. of7-oxo-7-desacetylamino-colchiceine were dissolved in 90 cc. of a 50%mixture of methylene chloride and methanol and the solution was cooledat C. Then, over a period of 10 minutes 180 cc. of a 1% diazomethanesolution in methylene chloride were added thereto. The resultingsolution was stirred for 1 hour at 0 C. and then a few drops of aceticacid were added and the solution was distilled to dryness under vacuum.The resulting product was taken up in 60 cc. of a mixture of methylenechloride and methanol (1:3) and after the methylene chloride wasdistilled off, the solution was left standing for 10 minutes to effectprecipitation. The precipitate was vacuum filtered and dried to obtain afirst crop of 3.78 gm. of 7-oxo-7-desacetylaminocolchicine which uponcrystallization from a mixture of methylene chloride and ethyl acetateyielded 2.93 gm. of 7-oxo-7-desacetylamino-colchicine having a meltingpoint of 230 C.

The product occurred in the form of small rectangular rods insoluble inwater, aqueous dilute acids and alkalis, ether and benzene, slightlysoluble in alcohols and soluble in acetone and chloroform.

Analysis (C H O Molecular weight=356.36. Calculated: C, 67.4%; H, 5.66%.Found: C, 67.1%; H, 5.7%.

This compound is not described in the literature.

By fractional crystallization of the methanol mother liquors,7-oxo-7-desacetylamino-iso-colchicine having a melting point of 190 C.was obtained. The said product occurred in the form of prisms insolublein water, dilute aqueous acids and alkalis, ether and benzene, slightlysoluble in alcohols and acetone and soluble in chloroform.

Analysis (C H O Molecular weight=356.36. Calculated: C, 67.40%; H,5.66%. Found: C, 67.6%; H 5.7%.

This product is not described in the literature.

EXAMPLE II Preparation of 7-oxo-7-desacetylamino-colchiceinamide 100 mg.of 7-oxo-7-desacetylamino-colchicine in cc. of methanol were reactedwith 2 cc. of concentrated aqueous ammonia for 24 hours while stirringat room temperature, chloroform was added to the solution, thechloroformic layer was washed with water, dried and distilled to drynessto obtain a residue of 7-oxo-7-desacetylamino-colchiceinamide.

This product is not described in the literature.

EXAMPLE 1H Preparation of the 7-oxo-7-desacetylamino-N-methyl-colchiceinamide 85 mg. of 7-oxo-7-desacetylamino-colchicine,dissolved in 25 cc. of methanol, were reacted with 25 cc. of an aqueoussolution of 36% monomethylamine. The mixture was allowed to stand whilestirring for a period of 18 hours at 20 C. and then was extracted withmethylene chloride. The extract as washed with water and distilled todryness. The residue was crystallized from a mixture of ethyl acetateand ether (2:3) to obtain 490 mg. (yield: 57%) of product having amelting point of 213 C. By recrystallization from a mixture ofmethylenechloride and methanol, 400 mg. of7-oxo-7-desacetylamino-N-methyl-colchiceinamide melting at 215 C. wereobtained.

The product Qccurred in the form of small yellow rods and hexagonalprisms insoluble in water, benzene and ether, slightly soluble inethanol and soluble in chlo roform and acetone.

Analysis (C H O N): Molecular weight=355.3 8. Calculated: C, 67.59%; H,5.96%; N, 3.94%. Found: C, 67.6%; H, 6.0%; N, 4.2%.

This compound is not described in the literature.

EXAMPLE IV Preparation of 7-oxo-7-desacetylamino-N-benzylcolchiceinamidemg. of 7-oxo-7-desacetylamino-colchicine in solution in 5 cc. ofmethanol, were reacted with 1 cc. of benzylamine. The reaction mixturewas allowed to stand at room temperature for 48 hours and thenchloroform was added. The chloroformic layer was washed with water, with0.1 N hydrochloric acid and with water, air dried and distilled todryness to obtain a residue of7-oxo-7-desacetylamino-N-benzyl-colchiceinamide.

This product is not described in the literature.

In an analogous fashion by reacting the appropriate amine with7-oxo-7-desacetylamino-colchicine, the following 7-oxo-7 desacetylaminocolchiceinamides have been prepared:

7-0xo-7-desacetylamino-N-ethyl-colchiceinamide;7-oxo-7-desacetylamino-N-propyl-colchiceinamide;7-oxo-7-desacetylamino-N-isopropyl-colchiceinamide;7-oxo-7-desacetylamino-N-butyl-colchiceinamide;7-oxo-7-desacetylamino-N,N-dimethyl-colchiceinamide;7-oxo-7-desacetylamino-N,N-diethyl-colchiceinamide;7-oxo-7-desacetylamino-N-methyl-N-ethyl-colchiceinamide;7-oxo-7-desacetylamino-N-methyl-N-benzyl-colchiceinamide; and7-oxo-7-desacetylamino-N-ethyl-N-isopropyl-colchiceinamide.

We claim:

1. 7-oxo-7-desacetylamino-colchicine.

2. 7-oxo-7-desacetylamino-colchiceine.

3. A process for the preparation of 7-oxo-7-desacetylamino-colchicinewhich comprises reacting N-desacetylcolchiceine withN-chloro-succinimide to form N-chloro- N-desacetyl-colchiceine,dehydrochlorinating the latter with an alkali metal hydroxide such asmethanolic potassium hydroxide to form7-imino-7-desacetylamino-colchiceine, reacting the latter with aqueousacetic acid to form 7-oxo-7-desacetylamino-colchiceine, reacting thelatter with diazomethane to form a mixture of 7-oxo-7-desacetylamino-colchicine and 7-oxo-7-desacetylaminoisocolchicine,separating the two components by fractional crystallization andrecovering 7-oxo-7-desacetylaminocolchicine.

4. A 7-oxo-7-desacetylamino-colchicinic compound of the formula CHaOCHaO O I CHaO wherein Z is lower alkoxy.

References Cited UNITED STATES PATENTS 3,393,240 7/1968 Muller et al.260-571 DANIEL D. HORWITZ, Primary Examiner.

US. or. X.R.

1960. THE INVENTION FURTHER RELATES TO NOVEL ANTI-MITOTIC COMPOSITIONSAND TO A METHOD OF MODIFYING CELL MITOSIS.